The acidic polysaccharide group known as glycosaminoglycans (GAG) including heparin/heparan sulfate (HS), chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronic acid attach to core proteins by means of covalent bonds, forming proteoglycans (PG) which are present in connective tissue and cell membranes. PG's, along with other cell-adhesive proteins, form the extracellular matrix, and are widely distributed in order to enable cells to live and to perform biological functions. In particular, heparan sulfate proteoglycans (HS-PG) are present in the tissue of almost all animals, and perform the extremely important functions of cell adhesion, morphogenesis and maintenance of functions.
Additionally, it has become clear that heparin/HS contained in PG's interacts with various types of cell growth factors and plays a considerable role in the control of cell differentiation and proliferation. Fibroblast growth factors (FGF) form the FGF family (currently, FGF1–FGF10 are known) having a high affinity with heparin/HS, and act with specificity with respect to vascular endothelial cells, Kaposi's sarcoma cells and epidermal keratinocytes. Such activity of FGF's is believed to occur as a result of binding specifically to FGF receptors (FGFR) on the cell surface. That is, as shown schematically in FIG. 1, heparin/HS pierces the membrane holds and preserves unstable FGF molecules in a stable state in the vicinity of the cell, and supports FGF bonds to the receptors (FGFR) on the cells as needed while protecting the FGF from proteolytic enzymes and oxidative decomposition. The FGF bonding to the FGFR causes the proliferation signal to be transmitted and promotes cell proliferation. This mechanism has been proven by much research which suggests that FGF's and FGFR's cannot bind without the presence of heparin/HS (for example, see M. Ishihara, Glycobiology, 4, 817–824 (1994)).
Heparin/HS is composed of a repeating structure of disaccharides including uronic acid having a carboxymethyl group and glucosamin having an acetyl group, and an important characteristic is the sulfation of hydroxyl groups and amino groups present in the molecule in various proportions. About 10 types of sulfation of the disaccharides have been identified, and heparin and HS are divided depending on differences in the sulfation. Additionally, cells are believed to control the activity of the FGF family by themselves preparing various types of heparin/HS of different levels of sulfation and molecular chain lengths according to their type and state.
Aside from controlling the activity of FGF, heparin/HS, which can take various sulfate structures as described above, interact with roughly 80% of cytokines which contribute to a wide range of biological reactions from cellular migration and proliferation to inflammatory reaction, with matrix adhesion molecules, metabolism-related substances and blood coagulation factors, thus performing an extreme variety of functions in the body. However, due to this multifunctionality, heparin/HS can oftentimes cause unwanted side effects when the native heparin/HS molecule is entirely used, thus restricting the use of heparin/HS in the field of pharmaceuticals and medicine.
On the other hand, the various functions of heparin/HS are known to change dramatically according to the molecular chain length. For example, while antithrombin III which inhibits blood coagulation binds with a characteristic structural domain having a 3-O-sulfate group contained in heparin/HS, a sequence of at least 5 saccharides is necessary to express this anti-coagulant activity, so that in actual practice, smaller molecules make reduced activity inevitable. Additionally, in order to ensure expression of FGF1 and FGF4 activity, a structural domain of at least 10 saccharides containing an abundance of 2-O-sulfate groups and 6-O-sulfate groups is necessary.
Recently, experiments have been performed to use the active domain of heparin/HS molecules as oxidatively fragmented heparinoids for the purpose of controlling only the cell growth factor activity among the various complexed functions of heparin/HS (M. Ishihara et al., J. Biol. Chem., 268, 4675–4683 (1993)). However, this research brought to light such problems as the control for the activity of various types of growth factors due to the heparinoid fragments being inadequate, and side effects such as higher bleeding tendencies due to increasing the concentration of heparinoids needed in order to maintain the desired activity.
Furthermore, in order to apply GAGs containing heparin/HS to various fields, they must be efficiently attached to hydrophobic resin products commonly used in the medical field such as polystyrenes and polycarbonates, but GAGs and their fragments generally have a high water solubility and are difficult to adsorb and attach firmly to the various resin products so that, for example, it is difficult to apply GAGs to diagnostic beads or culture dishes in order to make them useful for general basic medical research or clinical medicine.